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Human MRGPRX1 Stable Cell Line-CHO dhfr- Galphaqi5

Cat.No.: CSC-RG0353
Cell Line Description: CHO dhfr- Galphaqi5-HuMRGPRX1 cell line is clonally-derived from a CHO-K1 cell line,lacking the enzyme dihydrofolate reductase (DHFR), which has been transfected with a human MAS-related GPR, member X1 (MRGPRX1) to allow stably express of the human MRGPRX1. It is an example of a cell line transfected using our proprietary CBTGS gene screening and amplification system.
Background: MRGX1 (MAS-related GPR member X1) is also known as SNSR4 (sensory neuron-specific G-protein-coupled receptor 4). It can be potently activated by enkephalins including BAM22 and BAM(8-22). MRGX1 receptor is expressed solely in small diameter primary sensory neurons. This restricted expression pattern is of considerable therapeutic interest because small nociceptors transmit chronic pain messages.
Growth Properties: Adherent
Morphology: Epithelial-like
Host Cell: CHO dhfr- Galphaqi5
Cell Line Validation: 1. Gene expression: RT-PCR experiments determined specific expression of human MRGPRX1.2. Protein expression: MRGPRX1 expression in this cell line has been validated by WB.3. Functional validation: Functional assays.
Sub-type: Mas related
Propagation: Complete growth medium: Alpha-MEM, 10% FBS, 100μg/mL hygromycin, 800 μg/mL G418Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Starting Cells From Frozen Cell Stock: 1. Remove the packaging cell lines from liquid nitrogen and carry out a quick thaw. Float the cells in the 37°C water bath for 2 minutes until nearly (80%) thawed. Once cells are thawed, it is important to dilute the cells 1:10 in growth media immediately to reduce the potentially toxic effects of the DMSO preservative on the cells.2. Clean the outside of the vial with 70% ethanol before opening.3. Remove the cells from the vial and add slowly into a 15ml conical tube containing 10 ml pre-warmed media. 4. Centrifuge for 3 minutes 1000 xg to pellet cells and remove the supernatant.5. Add 14 ml of media and transfer cells to a T25 flask or a 100 mm culture dish.6. Place the cells in the 37°C incubator with 5% CO2.7. Allow incubation for 3-4 days to reach confluence. The cells will re-attach to the surface over a period of several days in culture at 37°C.
Subculturing: 1. Remove and discard culture medium.2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.3. Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.4. Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.5. Add appropriate aliquots of the cell suspension to new culture vessels.Incubate cultures at 37°C.Subcultivation Ratio: A subcultivation ratio of 1:4 to 1:6 is recommended Medium Renewal: 2 to 3 times per week
Mycoplasma: Mycoplasma Status: Negative (MycoAlert Kit)
Freeze Medium: Complete growth medium 90%; DMSO, 10%
Storage: Liquid nitrogen
Preservation: 1. Detach cells from culture dish according to the Sub-Culture Procedure.2. Resuspend cells at a density of 5 x 10^6 cells/mL in freeze medium.Note: A T-75 culture flask typically yields enough cells for preparing two frozen vials.3. Aliquot 1 mL cells into cryogenic vials.4. Place vials in a freezing container and store at –80 °C overnight.5. Transfer vials to liquid nitrogen for long term storage. If properly stored, cells should remain stable for years.
Safety Considerations: The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach.
Ship: Dry ice
Gene Name: MRGPRX1 MAS-related GPR, member X1 [ Homo sapiens ]
Official Symbol: MRGPRX1
Synonyms: MRGPRX1; MAS-related GPR, member X1; mas-related G-protein coupled receptor member X1; MRGX1; G protein-coupled receptor MRGX1; G protein-coupled receptor SNSR3; sensory neuron-specific G-protein coupled receptor 3/4; GPCR; SNSR4;
Gene ID: 259249
mRNA Refseq: NM_147199
Protein Refseq: NP_671732
MIM: 607227
UniProt ID: Q96LB2
Chromosome Location: 11p15.1
Function: G-protein coupled receptor activity; receptor activity; signal transducer activity;

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